Adenovirus E1A Oncogene Induces Rereplication of Cellular DNA and Alters DNA Replication Dynamics

The oncogenic property of the adenovirus (Ad) transforming E1A protein is linked to its capacity to induce cellular DNA synthesis which occurs as a result of its interaction with several host proteins, including pRb and p300/CBP. While the proteins that contribute to the forced induction of cellular DNA synthesis have been intensively studied, the nature of the cellular DNA replication that is induced by E1A in quiescent cells is not well understood. Here we show that E1A expression in quiescent cells leads to massive cellular DNA rereplication in late S phase. Using a single-molecule DNA fiber assay, we studied the cellular DNA replication dynamics in E1A-expressing cells. Our studies show that the DNA replication pattern is dramatically altered in E1A-expressing cells, with increased replicon length, fork velocity, and interorigin distance. The interorigin distance increased by about 3-fold, suggesting that fewer DNA replication origins are used in E1A-expressing cells. These aberrant replication events led to replication stress, as evidenced by the activation of the DNA damage response. In earlier studies, we showed that E1A induces c-Myc as a result of E1A binding to p300. Using an antisense c-Myc to block c-Myc expression, our results indicate that induction of c-Myc in E1A-expressing cells contributes to the induction of host DNA replication. Together, our results suggest that the E1A oncogene-induced cellular DNA replication stress is due to dramatically altered cellular replication events and that E1A-induced c-Myc may contribute to these events.     

Seric factors influence cell cycle modifications during commitment of murine erythroleukemia cells to differentiation.

Cell cycle modifications are among the early events which take place during the induced differentiation of murine erythroleukemia (MEL) cells; a transient accumulation of the cells in the G1 phase of the cell cycle, followed by a re-entry of the cells into a proliferation state, has been described. In order to characterize a putative role of serum in such variations, we have studied the modifications of the cell cycle parameters when cells were induced to differentiate in the presence or in the absence of seric factors. We show that, in the absence of exogenous factors brought by serum, the G1 accumulation was enhanced both in amplitude and in duration, but cells were still able to bypass the G1 block and re-enter into the S phase. These results indicate that the resumption of cell proliferation after the transient block is under synergistic control of seric and endogenous factors, but these later are sufficient to overcome the block. However, MEL cells were unable to differentiate in the absence of seric factors, as measured by the number of benzidine-positive cells during induction with hexamethylene-bisacetamide (HMBA) or butyric acid. This capacity to differentiate was recovered when serum was added back to the culture medium, and the efficiency of recovery was maximal when cells underwent a full round of DNA replication in the presence of serum after the G1 block. The analysis of two molecular markers of cell differentiation confirmed these results.     

Alterations in cell nuclei during apoptosis

Apoptosis is a genetically programmed phenomenon that aids in maintaining homeostasis in multicellular organisms. The characteristic morphological features of apoptosis are highly conservative and are dependent on the cell type and the apoptotic inducer. The nuclear events occurring during apoptosis include changes at the molecular level (i.e. DNA cleavage, modifications of nuclear polypeptides, and proteolysis of several proteins important for cell maintenance), and, consequently, alterations at the morphological level (i.e. chromatin condensation, nuclear shrinkage, DNA fragmentation and apoptotic body formation). These events are still not fully understood. It is very probable that a progressive decrease in pH could also be an essential factor for the induction of nuclease and protease activities, and an important element of the optimal conditions for their function. This review details the current state of knowledge on apoptotic nuclear events, with particular focus on the proteins involved in the execution of apoptosis in cell nuclei, and on the differences in substrate cleavage profiles for different types of cell undergoing cell death.     

Metallothionein gene expression is regulated by serum factors and activators of protein kinase C.

The exact physiological role of metallothionein (MT) is not clear. It has been suggested that these low-molecular-weight, highly inducible, heavy-metal-binding proteins serve in the regulation of intracellular Zn metabolism. Among the Zn-requiring systems are several enzymes involved in DNA replication and repair. Therefore, during periods of active DNA synthesis there is likely to be an increased demand for Zn, which could be met by elevated MT synthesis. For that reason, we examined whether stimulation of cellular proliferation leads to increased expression of MT. We report here that treatment of cultured mammalian cells with serum growth factors and activators of protein kinase C, all of which are known to have growth stimulatory activity, led to induction of MT mRNA. One of the required steps in the signal transduction pathways triggered by these agents, ending in MT induction, appears to be the activation of protein kinase C.     

The histone methyltransferase SET8 is required for S-phase progression

Chromatin structure and function is influenced by histone posttranslational modifications. SET8 (also known as PR-Set7 and SETD8) is a histone methyltransferase that monomethylates histonfe H4-K20. However, a function for SET8 in mammalian cell proliferation has not been determined. We show that small interfering RNA inhibition of SET8 expression leads to decreased cell proliferation and accumulation of cells in S phase. This is accompanied by DNA double-strand break (DSB) induction and recruitment of the DNA repair proteins replication protein A, Rad51, and 53BP1 to damaged regions. SET8 depletion causes DNA damage specifically during replication, which induces a Chk1-mediated S-phase checkpoint. Furthermore, we find that SET8 interacts with proliferating cell nuclear antigen through a conserved motif, and SET8 is required for DNA replication fork progression. Finally, codepletion of Rad51, an important homologous recombination repair protein, abrogates the DNA damage after SET8 depletion. Overall, we show that SET8 is essential for genomic stability in mammalian cells and that decreased expression of SET8 results in DNA damage and Chk1-dependent S-phase arrest.     

Chromatin architecture and functions: the role(s) of poly(ADP-RIBOSE) polymerase and poly(ADPribosyl)ation of nuclear proteins.

Epigenetic states that allow chromatin fidelity inheritance can be mediated by several factors. One of them, histone variants and their modifications (including acetylation, methylation, phosphorylation, poly(ADP-ribosyl)ation, and ubiquitylation) create distinct patterns of signals read by other proteins, and are strictly related to chromatin remodelling, which is necessary for the specific expression of a gene, and for DNA repair, recombination, and replication. In the framework of chromatin-controlling factors, the poly(ADP-ribosyl)ation of nuclear proteins, catalysed by poly(ADP-ribose)polymerases (PARPs), has been implicated in the regulation of both physiological and pathological events (gene expression/amplification, cellular division/differentiation, DNA replication, malignant transformation, and apoptotic cell death). The involvement of PARPs in this scenario has raised doubts about the epigenetic value of poly(ADP-ribosyl)ation, because it is generally activated after DNA damage. However, one emerging view suggests that both the product of this reaction, poly(ADP-ribose), and PARPs, particularly PARP 1, play a fundamental role in recruiting protein targets to specific sites and (or) in interacting physically with structural and regulatory factors, through highly reproducible and inheritable mechanisms, often independent of DNA breaks. The interplay of PARPs with protein factors, and the combinatorial effect of poly(ADPribosyl)ation with other post-translational modifications has shed new light on the potential and versatility of this dynamic reaction.     

Regulation of the DNA replication fork: a way to fight genomic instability

DNA replication is a complex mechanism that functions due to the coordinated interplay of many factors. In the last few years, numerous studies have suggested that DNA replication factors are closely implicated in several DNA transaction events that maintain the integrity of the genome. Therefore, DNA replication fork factors have to be considered as part of a general process that aims to protect and replicate the genome in order to allow correct functioning of a cell and its eventual daughter cells. This is illustrated by the numerous factors that have a well-defined function at the DNA replication fork, but also play crucial roles in different DNA repair pathways such as base excision repair, nucleotide excision repair, double-strand break repair, and mismatch repair. Moreover, several of the replisome proteins have also been shown to be essential in sensing and transducing DNA damages through the checkpoint cascade pathways, including the recently characterised alternative clamps and clamp-loaders. In this review we present DNA replication factors that are involved in different DNA transaction and checkpoint regulation pathways, with emphasis on the link between DNA replication and maintenance of genomic stability.     

The mechanism of sensitivity to phleomycin in growing Escherichia coli cells.

Stationary-phase Escherichia coli B cells transferred to new growth medium are initially resistant to net DNA breakage by low concentrations of phleomycin, and become sensitive as DNA replication commences. From studies with inhibitors of various stages of the DNA replication cycle it is evident that it is not DNA synthesis itself that is required for induction of DNA breakage by phleomycin, but events associated with the initiation of DNA replication. Termination of replication in the absence of further initiaiton results in resistance to phleomycin. The cellular change responsible for changes in sensitivity to phleomycin could be the attachment of the bacterial chromosome to the cell membrane at initiation and detachment on termination of replication, suggesting an alteration in the balance between cellular DNA breakage and repair processes for membrane-associated compared with non-membrane-associated DNA.     

Effects of sodium butyrate,a new pharmacological agent,on cells in culture

Summary Sodium butyrate, at millimolar concentrations, when added to cell cultures produces many morphological and biochemical modifications in a reversible manner. Some of them occur in all cell lines. They concern regulatory mechanisms of gene expression and cell growth: an hyperacetylation of histone resulting from an inhibition of histone deacetylase and an arrest of cell proliferation are almost constantly observed. Some other modifications vary from one cell type to another: induction of proteins, including enzymes, hormones, hemoglobin, inhibition of cell differentiation, reversion of transformed characteristics of cells to normal morphological and biochemical pattern, increase in interferon antiviral efficiency and induction of integrated viruses. Most if not all these effects of butyrate could result from histone hyperacetylation, from changes in chromatin structures as measured by accessibility to DNases and from modifications in cytoskeleton assembly. We do not know at the present time whether butyrate acts on a very specific target site in cell or if it acts on several cell components.     

Effects of sodium butyrate, a new pharmacological agent, on cells in culture

Sodium butyrate, at millimolar concentrations, when added to cell cultures produces many morphological and biochemical modifications in a reversible manner. Some of them occur in all cell lines. They concern regulatory mechanisms of gene expression and cell growth: an hyperacetylation of histone resulting from an inhibition of histone deacetylase and an arrest of cell proliferation are almost constantly observed. Some other modifications vary from one cell type to another: induction of proteins, including enzymes, hormones, hemoglobin, inhibition of cell differentiation, reversion of transformed characteristics of cells to normal morphological and biochemical pattern, increase in interferon antiviral efficiency and induction of integrated viruses. Most if not all these effects of butyrate could result from histone hyperacetylation, from changes in chromatin structures as measured by accessibility to DNases and from modifications in cytoskeleton assembly. We do not know at the present time whether butyrate acts on a very specific target site in cell or if it acts on several cell components.     

Proliferating cell nuclear antigen: More than a clamp for DNA polymerases

DNA metabolic events such as replication, repair and recombination require the concerted action of several enzymes and cofactors. Nature has provided a set of proteins that support DNA polymerases in performing processive, accurate and rapid DNA synthesis. Two of them, the proliferating cell nuclear antigen and its adapter protein replication factor C, cooperate to form a moving platform that was initially thought of only as an anchor point for DNA polymerases δ and ε. It now appears that proliferating cell nuclear antigen is also a communication point between a variety of important cellular processes including cell cycle control, DNA replication, nucleotide excision repair, post-replication mismatch repair, base excision repair and at least one apoptotic pathway. The dynamic movement of proliferating cell nuclear antigen on and off the DNA renders this protein an ideal communicator for a variety of proteins that are essential for DNA metabolic events in eukaryotic cells.     

Identification of novel factors involved in or regulating initiation of DNA replication by a genome-wide phenotypic screen in Saccharomyces cerevisiae

DNA replication in eukaryotic cells is tightly regulated to ensure faithful inheritance of the genetic material. While the replicators, replication origins and many replication-initiation proteins in Saccharomyces cerevisiae have been identified and extensively studied, the detailed mechanism that controls the initiation of DNA replication is still not well understood. It is likely that some factors involved in or regulating the initiation of DNA replication have not been discovered. To identify novel DNA replication-initiation proteins and their regulators, we developed a sensitive and comprehensive phenotypic screen by combining several established genetic strategies including plasmid loss assays with plasmids containing a single versus multiple replication origins and colony color sectoring assays. We isolated dozen of mutants in previously known initiation proteins and identified several novel factors, including Ctf1p Ctf3p, Ctf4p, Ctf18p, Adk1p and Cdc60p, whose mutants lose plasmid containing a single replication origin at high rates but lose plasmid carrying multiple replication origins at lower rates. We also show that overexpression of replication initiation proteins causes synthetic dosage lethality or growth defects in ctf1 and ctf18 mutants and that Ctf1p and Ctf18p physically interact with ORC, Cdt1p and MCM proteins. Furthermore, depletion of both Ctf1p and Ctf18p prevents S phase entry, retards S phase progression, and reduces pre-RC formation during the M-to-G1 transition. These data suggest that Ctf1p and Ctf18p together play important roles in regulating the initiation of DNA replication.     

Dynamic targeting of the replication machinery to sites of DNA damage

Components of the DNA replication machinery localize into discrete subnuclear foci after DNA damage, where they play requisite functions in repair processes. Here, we find that the replication factors proliferating cell nuclear antigen (PCNA) and RPAp34 dynamically exchange at these repair foci with discrete kinetics, and this behavior is distinct from kinetics during DNA replication. Posttranslational modification is hypothesized to target specific proteins for repair, and we find that accumulation and stability of PCNA at sites of damage requires monoubiquitination. Contrary to the popular notion that phosphorylation on the NH2 terminus of RPAp34 directs the protein for repair, we demonstrate that phosphorylation by DNA-dependent protein kinase enhances RPAp34 turnover at repair foci. Together, these findings support a dynamic exchange model in which multiple repair factors regulated by specific modifications have access to and rapidly turn over at sites of DNA damage.     

Cell Cycle-Regulated Protein Abundance Changes in Synchronously Proliferating HeLa Cells Include Regulation of Pre-mRNA Splicing Proteins

Cell proliferation involves dramatic changes in DNA metabolism and cell division, and control of DNA replication, mitosis, and cytokinesis have received the greatest attention in the cell cycle field. To catalogue a wider range of cell cycle-regulated processes, we employed quantitative proteomics of synchronized HeLa cells. We quantified changes in protein abundance as cells actively progress from G1 to S phase and from S to G2 phase. We also describe a cohort of proteins whose abundance changes in response to pharmacological inhibition of the proteasome. Our analysis reveals not only the expected changes in proteins required for DNA replication and mitosis but also cell cycle-associated changes in proteins required for biological processes not known to be cell-cycle regulated. For example, many pre-mRNA alternative splicing proteins are down-regulated in S phase. Comparison of this dataset to several other proteomic datasets sheds light on global mechanisms of cell cycle phase transitions and underscores the importance of both phosphorylation and ubiquitination in cell cycle changes.     

Loss of proliferative capacity and induction of senescence in oxidatively stressed human fibroblasts

Cellular senescence can result from short, dysfunctional telomeres, oxidative stress, or oncogene expression, and may contribute to aging. To investigate the role of cellular senescence in aging it is necessary to define the time-dependent molecular events by which it is characterized. Here we investigated changes in levels of key proteins involved in cell cycle regulation, DNA replication, and stress resistance in senescing human fibroblasts following oxidative stress. An immediate response in stressed cells was dephosphorylation of retinoblastoma (Rb) and cessation of DNA synthesis. This was followed by sequential induction of p53, p21, and p16. Increase in hypophosphorylated Rb and induction of p53 and p21 by a single stress treatment was transient, whereas sustained induction or dephosphorylation were achieved by a second stress. Down-regulation of the critical DNA replication initiation factor Cdc6 occurred early after stress concurring with p53 induction, and was followed by a decrease in Mcm2 levels. A late event in the stress-induced molecular sequence was the induction of SOD1, catalase, and HSP27 coinciding with development of the fully senescent phenotype. Our data suggest that loss of proliferative capacity in oxidatively stressed cells is a multistep process regulated by time-dependent molecular events that may play differential roles in induction and maintenance of cellular senescence.     

酵母模式生物研究表观遗传调控基因组稳定性的进展

基因组的遗传稳定性是维持正常的细胞复制、增殖和分化的关键。外源因素和内源因素造成的DNA损伤及其修复失败, 是各种遗传疾病发生的根本原因。表观遗传调控(包括DNA甲基化、组蛋白修饰和非编码RNA)在DNA损伤修复和细胞周期调控方面发挥着重要的作用, 也是维持基因组稳定性的基础。酵母作为单细胞真核生物, 是最早开展表观遗传学研究的物种之一, 特别是在DNA损伤修复和异染色质形成等方面的研究, 为揭示遗传稳定性的本质提供了理论依据。国际上前期以酵母为模式生物研究表观遗传学的报道主要集中于组蛋白修饰领域; 近期利用裂殖酵母作为模式生物研究RNAi指导的组蛋白修饰也有了一定的进展。文章以酵母作为模式生物, 论述了表观遗传修饰在维持基因组遗传稳定性中的研究进展、作用机制和今后的发展趋势。